This project is designed to purify and characterize the rate-limiting enzymes involved in the synthesis of GABA, glycine and taurine, e.g., L-glutamate decarboxylase (GAD), serine transhydroxymethylase (serine-T), and cysteic acid decarboxylase (CAD), respectively, and to elucidate the role of these enzymes in the regulation of the effective level of GABA, glycine and taurine in connection with their important roles in the mammalian central nervous system. The purified enzyme preparations also will serve as antigens for the production of specific antibodies, so that the precise cellular and subcellular locations of these enzymes can be visualized by immunocytochemical method. More specifically, I plan to perform the following studies: (1) Purify various forms of GAD, serine-T, and CAD by methods similar to those we have developed for the successful purification of GAD and GABA-transaminase (GABA-T) from mouse brain and GAD from beef heart, with necessary modifications. (2) Perform immunochemical studies with a view to determining species-, tissue- and cell-specificities of these enzymes from different sources. (3) Localize these enzymes on tissue section at light and electron microscopic levels by methods similar to those we had successfully developed for the visualization of GAD in various regions of rat central nervous system. (4) Perform physical, chemical and enzymatic studies of the native enzymes and their subunits with special emphasis on the regulatory mechanism of the enzyme activities and the detailed structure of active site and binding sites with a view to designing specific inhibitors for pharmacological applications. (5) Perform developmental studies of these enzymes with a view to correlating biochemical, immunochemical and morphological changes during maturation. Recent progress in GABA-nergic pathway resulting from the work of the Principal Investigator on purification and characterization of GAD and GABA-T shows the feasibility of the proposed studies.